Cerebellar development relies on the coordinated proliferation and differentiation of progenitors from the ventricular zone (VZ) and rhombic lip (RL). To systematically map their spatiotemporal dynamics, we performed EdU pulse labeling by injecting pregnant mice with EdU and collecting embryonic cerebella at daily intervals over five consecutive days as well as an acute half-an-hour post EdU injection. EdU labeling identifies actively dividing progenitor cells at the time of injection. As development progresses, EdU+ cells can be tracked to study their differentiation and migration, revealing the temporal dynamics of VZ and RL progenitor-derived neurons in the cerebellum. Using multiplex immunohistochemistry with VZ- and RL-derived cell-type specific markers, we tracked the spatial distribution and differentiation of EdU-labeled cells, distinguishing VZ- and RL-derived progenitor lineages. Additionally, we outline a strategy to isolate EdU+ cells for single-cell RNA sequencing (scRNA-seq) and ATAC sequencing (ATAC-seq), enabling a comprehensive molecular characterization of progenitor fate transitions. This approach provides a high-resolution developmental trajectory of cerebellar progenitors, offering new insights into the regulatory mechanisms driving cerebellar neurogenesis and their disruptions in neurodevelopmental disorders.

Acomys Cahirinus (spiny mice) are remarkable creatures that exhibit key differences in inflammatory response, regeneration, and aging compared to mice. Adult neurogenesis - the production of new neurons- in the hippocampal niche declines with age in most mammals, yet Acomys exhibits sustained neurogenic potential, presenting a unique model for regenerative neuroscience. This study leverages advanced image analysis software (Imaris) to develop robust pipelines for quantifying neural stem cell (NSC) and intermediate progenitor (IP) proliferation and fate determination in Acomys versus standard laboratory mice (Mus musculus). Using EdU incorporation to track S-phase entry and a 4D pulse-labeling approach, we assess neurogenic niche activity across species. Additionally, we extend this analysis to aging Acomys, utilizing consistent sectioning, staining, and imaging parameters to confirm continuous progenitor proliferation in young and old cohorts. Our findings provide critical insights into the cellular and molecular mechanisms underlying sustained neurogenesis in Acomys, offering prospective therapeutic targets for age-related neurodegenerative conditions.

Understanding the dynamic behaviors of cells in the developing human brain is essential for elucidating the mechanisms that drive both normal and abnormal neurodevelopment. Using lentiviruses encoding fluorescent proteins, we infected cells in slices from different regions of the developing human cerebellum to track their movements over several hours. We then captured timelapse images of these fluorescent slices under a microscope, allowing us to visualize their dynamic behavior. Using live imaging analysis software, hundreds of individual cells were then tracked and characterized. Our analysis found several key processes, including novel modes of cell division and differentiation, neuronal migration, and intercellular communication. This approach allowed us to map a timeline of critical events that shape cerebellar architecture. This research aims to help us gain insight into neurodevelopmental disorders, where disturbances in fundamental biological processes underlie disease progression.

The cerebellar ventricular zone (VZ) is the primary source of progenitor cells that give rise to all cerebellar GABAergic neurons, including Purkinje cells (PCs) and interneurons (INs). While the VZ has been well studied in mice, much less is known about its role in human brain development. In this study, we investigated how progenitors and neurons form in the human cerebellar VZ, using in situ hybridization, immunohistochemistry, and single-cell RNAseq analysis. Our findings reveal several key differences from the mouse model. We found that Purkinje cells are generated during a brief two-week period, even before the cerebral cortex begins to develop. Interneurons, on the other hand, start differentiating a few weeks later and mature on a timescale of months to years. A unique feature of human cerebellar development is the presence of specialized inner and outer subventricular zones (SVZ), which are absent in mice. Most differentiation occurs in these regions, with the first wave taking place in the outer SVZ. Additionally, we observed variations in Purkinje cell arrangement and number, including a subset of Purkinje cells that continue expressing cell cycle genes, suggesting a more complex and prolonged developmental profile compared to mice. By characterizing these developmental processes, our study provides new insights into human cerebellar development, highlighting important structural and temporal differences from animal models. These findings may have implications for understanding neurodevelopmental disorders.